The cardiac cycle is a tightly regulated process in which the heart generates power during systole and then relaxes during diastole. Dysfunction of this cycle, as seen in patients with cardiomyopathies, can lead to heart failure and arrhythmias. Familial cardiomyopathies, found in 1:500 members of the population, are caused primarily by mutation of sarcomeric proteins involved in muscle contraction. It has been proposed that these mutations affect the regulation of the cardiac cycle and the ability of the heart to generate the force and power necessary to effectively pump blood to the body, leading to restructuring of the heart tissue and eventual heart failure. Despite many elegant experiments, it is not well understood how mutations on the molecular scale lead to changes in cellular contractility and tissue organization. This research will help to bridge this major gap in our understanding of the disease pathogenesis. By combining single molecule, ensemble kinetic, stem cell engineering, and tissue engineering techniques, our approach will allow us to understand how molecular changes in contractility associated with cardiomyopathy mutations lead to alterations in force and power output in human cardiac tissue. During the K99 portion of the grant, we will develop (1) a novel single-molecule in vitro assay that will enable us to mimic working conditions in the heart and (2) human cardiac microtissues in microelectromechanical devices that will enable us to examine the contractile and structural properties of human cardiac tissue. We will use assays to study the factors that regulate force and power output at the molecular, cellular, and tissue levels in healthy hearts. Importantly, the techniques and approaches developed in the K99 portion of the grant will complement the PI's current skill set, enabling him to establish an innovative research program. During the R00 portion of the grant, we will apply these newly developed technologies to studying how mutations associated with human cardiomyopathies lead to alterations in force and power output at the molecular, cellular, and tissue levels. This multi-tiered approach will give us an unprecedented understanding of the disease pathogenesis. Moreover, the techniques and approaches developed during the funding period should open several new avenues for future studies as the PI transitions to being a fully- independent scientist.